This invention relates generally to the field of growth factors and specifically to fragments of Connective Tissue Growth Factor (CTGF) and methods of use thereof.
Growth Factors. Growth factors can be broadly defined as multifunctional, locally acting intracellular signaling polypeptides which control both the ontogeny and maintenance of tissue form and function. The protein products of many proto-oncogenes have been identified as growth factors and growth factor receptors.
Growth factors generally stimulate target cells to proliferate, differentiate and organize in developing tissues. The action of growth factors is dependent on their binding to specific receptors that stimulate a signaling event within the cell. Examples of growth factors include platelet derived growth factor (PDGF), insulin like growth factor (IGF), transforming growth factor beta (TGF-xcex2), transforming growth factor alpha (TGF-xcex1), epidermal growth factor (EGF) and connective tissue growth factor (CTGF). Each of these growth factors has been reported to stimulate cells to proliferate.
Connective Tissue Growth Factor. CTGF is a cysteine rich monomeric peptide a molecular weight of about 38 kd. As-previously reported, CTGF has both mitogenic and chemotactic activities for connective tissue cells. CTGF is secreted by cells and is believed to be active upon interaction with a specific cell receptor.
CTGF is a member of a family of growth regulators which include, for example, mouse (fisp-12) and human CTGF, Cyr61 (mouse), Cef10 (chicken), and Nov (chicken). Based on sequence comparisons, is has been suggested that the members of this family have a modular structure consisting typically of at least one of the following: (1) an insulin-like growth factor domain responsible for binding; (2) a von Willebrand factor domain responsible for complex formation; (3) a thrombospondin type I repeat, possibly responsible for binding matrix molecules; and (4) a C-terminal module found in matrix proteins, postulated to be responsible for receptor binding.
The sequence of the cDNA for human CGTF contains an open reading frame of 1047 nucleotides with an initiation site at about nucleotide 130 and a TGA termination site at about nucleotide 1177, and encodes a peptide of 349 amino acids. The cDNA sequence for human CTGF has been previously disclosed in U.S. Pat. No. 5,408,040.
The CTGF open reading frame encodes a polypeptide which contains 39 cysteine residues, indicating a protein with multiple intramolecular disulfide bonds. The amino terminus of the peptide contains a hydrophobic signal sequence indicative of a secreted protein and there are two N-linked glycosylation sites at asparagine residues 28 and 225 in the amino acid sequence.
The synthesis and secretion of CTGF are believed to be selectively induced by TGF-xcex2 and BMP-2, as well as potentially by other members of the TGF-xcex2 superfamily of proteins. As reported in the art, although TGF-xcex2 can stimulate the growth of normal fibroblasts in soft agar, CTGF alone cannot induce this property in fibroblasts. However, it has been shown that the synthesis and action of CTGF are essential for the TGF-xcex2 to stimulate anchorage independent fibroblast growth. See, e.g., Kothapalli et al., 1997, Cell Growth and Differentation 8(1):61-68 and Boes et al., 1999, Endocrinology 140(4):1575-1580.
With respect to biological activity, CTGF has been reported to be primarily mitogenic in nature (able to stimulate target cells to proliferate). CTGF has also been reported to have chemotactic activity. Pathologically, the full-length CTGF molecule has been reported to be involved in conditions where there is an overgrowth of connective tissue cells and overdeposition of the extracellular matrix. CTGF has also been described in the art to be associated with conditions relating to vascular endothelial cell migration and proliferation, and neovascularization. The diseases and disorders relating to these conditions, include, for example, fibrosis of the skin and major organs, cancer, and related diseases and disorders such as systemic sclerosis, angiogenesis, atherosclerosis, diabetic nephropathy, and renal hypertension. (See, e.g., Toshifurni et al., 1999, Journal of Cellular Physiology 18191):153-159; Shimo et al., 1999, Journal of Biochemistry 126(1):137-145; Murphy et al., 1999, Journal of Biological Chemistry 274(9):5830-5834; Wenger et al., 1999, Oncogene 18(4):1073-1080; Frzier et al., 1997, International Journal of Biochemistry and Cell Biology 29(1); 153-161; Oemar et al., 1997, Circulation 95(4);831-839.)
CTGF has also been reported to be useful in wound healing and repair of connective tissue, bone and cartilage. In this aspect, CTGF has been described as an inducer of bone, tissue, or cartilage formation in disorders such as osteoporosis, osteoarthritis or osteochondrytis, arthritis, skeletal disorders, hypertrophic scars, burns, vascular hypertrophy or wound healing. (See, e.g., U.S. Pat. No. 5,837,258; Ohnishi et al., 1998, Journal of Molecular and Cellular Cardiology 30(11):2411-2422; Nakanishi et al., 1997, Biochemical and Biophysical Research Communications 234(1):206-210; Pawar et al., 1995, Journal of Cellular Physiology 165(3):556-565.)
In summary, CTGF has been implicated in numerous fibrotic and cancerous conditions, and has been described to contribute to wound healing. As a result, there is a need in the art to identify useful-methods of modulating the activity of CTGF to treat these various diseases and disorders. Prior to the present invention, there has been no report that regions or domains of CTGF are responsible for signaling different biological activities. Moreover, prior to the instant invention, there has been no disclosure of treating diseases and disorders associated with cell proliferation and/or the overproduction of the extracellular matrix by inhibiting the biological activity of a specific region or domain of CTGF.
The present invention provides novel compositions and methods for the treatment of CTGF-associated diseases, disorders or ailments wherein the deposition of the extracellular matrix is implicated, including, for example, the induction of collagen synthesis and myofibroblast differentiation. More specifically, the compositions of the present invention comprise CTGF fragments comprising the N-terminal region of CTGF. In one aspect, the fragment of the present invention comprises at least a part of exons 2 or 3, or the polypeptide encoded thereby, is not the CTGF fragment disclosed in Brigstock et al., 1997, J. Biol. Chem. 272(32):20275-82, and further possesses the ability to induce synthesis of the extracellular matrix, including, but not limited to, the ability to induce collagen, and myfibroblast differentiation. In a further aspect, the fragment of the present invention comprises between about one-quarter and one-half the length of the full-length CTGF protein.
In one aspect, a fragment of connective tissue growth factor (CTGF) polypeptide having the activities as described above is provided. A fragment of the invention includes CTGF having an amino acid sequence encoded by at least exon 2 as set forth in FIG. 3. A fragment may also include an amino acid sequence encoded by at least exon 3 as set forth in FIG. 3. Further, a CTGF fragment of the invention may include an amino acid sequence encoded by at least exons 2 and 3 as set forth in FIG. 3. The invention also provides polynucleotide sequences encoding such fragments.
The present invention further comprises methods of using the CTGF fragments of the disclosed invention to identify compositions which can modulate the activity of said CTGF fragments, wherein such compositions may be used to control normal depostion of the extracellular matrix, such as collagen deposition, as desired. More specifically, the CTGF fragments may be used to identify compositions that may control normal deposition of collagen deposition and myfibroblast differentiation, wherein such composition may be used to inhibit, suppress, or increase the activity of the CTGF fragments of the present invention.
The compositions of the claimed invention further comprise CTGF modulators, for example, antibodies, antisense molecules, small molecules, and other compounds identified by the above methods, which can modulate the activity of the CTGF fragments of the present invention. In one aspect, the present invention provides CTGF modulators that inhibit or suppress the activity of CTGF or the CTGF fragments. In another aspect of the present invention, the CTGF modulators increase the activity of CTGF or the CTGF fragments, for example, in indications wherein the induction of CTGF activity is desirable, for example, in wound healing, tissue repair, and bone repair.
In another aspect of the invention, the methods of the present invention comprise the administration of an effective amount of the CTGF fragment modulators, alone or in combination with one or more compounds, to a patient in need to treat diseases, disorders or ailments wherein the manipulation of collagen synthesis is desired. More particularly, the methods of the instant invention are directed to utilizing the compounds capable of modulating the activity of the CTGF fragments of this invention to modulate collagen synthesis, and consequently, treat disorders related to the overabundance of collagen synthesis, including fibrotic disorders. Preferably, the disorders are of the dermis, the major organ and disorders related to the overproduction of scar tissue.
The present invention also provides pharmaceutical compositions comprising the CTGF fragments of the present invention. Such compositions may be useful in wound healing, bone and tissue repair, wherein the increased activity of CTGF is desirable.